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Conformational Mechanics, Adsorption, and Normal Force Interactions of Lubricin and Hyaluronic Acid on Model Surfaces† Debby P. Chang,‡,§,O Nehal I. Abu-Lail,#,O Farshid Guilak,|,⊥ Gregory D. Jay,∇ and Stefan Zauscher*,‡,§,| Department of Mechanical Engineering and Materials Science, Center for Biologically Inspired Materials and Material Systems, and Center for Biomolecular and Tissue Engineering, Duke UniVersity, Durham, North Carolina 27708, Department of Surgery, Duke UniVersity Medical Center, Durham North Carolina 27710, School of Chemical Engineering and Bioengineering, Washington State UniVersity, Pullman, Washington 99164, and Department of Emergency Medicine and DiVision of Engineering, Brown UniVersity, ProVidence, Rhode Island 02912 ReceiVed August 1, 2007. In Final Form: October 31, 2007 Glycoproteins, such as lubricin, and hyaluronic acid (HA) play a prominent role in the boundary lubrication mechanism in diarthrodial joints. Although many studies have tried to elucidate the lubrication mechanisms of articular cartilage, the molecular details of how lubricin and HA interact with cartilage surfaces and mediate their interaction still remain poorly understood. Here we used model substrates, functionalized with self-assembled monolayers terminating in hydroxyl or methyl groups, (1) to determine the effect of surface chemistry on lubricin and HA adsorption using surface plasmon resonance (SPR) and (2) to study normal force interactions between these surfaces as a function of lubricin and HA concentration using colloidal probe microscopy. We found that lubricin is amphiphilic and adsorbed strongly onto both methyl- and hydroxyl-terminated surfaces. On hydrophobic surfaces, lubricin likely adopts a compact, looplike conformation in which its hydrophobic domains at the N and C termini serve as surface anchors. On hydrophilic surfaces, lubricin likely adsorbs anywhere along its hydrophilic central domain and adopts, with increasing solution concentration, an extended tail-like conformation. Overall, lubricin develops strong repulsive interactions when compressing two surfaces into contact. Furthermore, upon surface separation, adhesion occurs between the surfaces as a result of molecular bridging and chain disentanglement. This behavior is in contrast to that of HA, which does not adsorb appreciably on either of the model surfaces and does not develop significant repulsive interactions. Adhesive forces, particularly between the hydrophobic surfaces, are large and not appreciably affected by HA. For a mixture of lubricin and HA, we observed slightly larger adsorptions and repulsions than those found for lubricin alone. Our experiments suggest that this interaction depends on unspecific physical rather than chemical interactions between lubricin and HA. We speculate that in mediating interactions at the cartilage surface, an important role of lubricin, possibly in conjunction with HA, is one of providing a protective coating on cartilage surfaces that maintains the contacting surfaces in a sterically repulsive state.
Introduction Under normal circumstances, diarthrodial joints in the body enable locomotion and activity while withstanding millions of loading cycles supporting several times the body weight.1,2 This remarkable function is attributed to the unique structure and composition responsible for the mechanical properties of the cartilage extracellular matrix. Articular cartilage is a thin, soft tissue that covers the bony ends of diarthrodial joints3 and presents an efficient lubricating surface for joint contact.4,5 Articular cartilage has only a limited capacity for self-repair,6 and damage
to this tissue can lead to degenerative pathological changes in the joint and joint diseases such as osteoarthritis (OA). A range of lubrication theories have been proposed over the years to describe friction in articular joints.1,7-13 Although the mechanisms behind joint lubrication remain controversial, there is general agreement that boundary lubrication plays an important role in joints under high load at low sliding speeds.1,8 At present, three types of boundary lubricants are considered to be involved, singly or in combination, in mediating friction in articular joints: (1) hyaluronan,14 (2) surface-active lipids,15-17 and (3) mucinous glycoproteins (lubricin).4,18-27 Boundary lubricants can affect
†
Part of the Molecular and Surface Forces special issue. * Corresponding author. E-mail:
[email protected]. Phone: (919) 6605360. Fax: (919) 660-5409. ‡ Department of Mechanical Engineering and Materials Science, Duke University. § Center for Biologically Inspired Materials and Material Systems, Duke University. | Center for Biomolecular and Tissue Engineering, Duke University. ⊥ Department of Surgery, Duke University Medical Center. # Washington State University. ∇ Brown University. O These authors contributed equally to this work. (1) Mow, V. C.; Ratcliffe, A.; Poole, A. R. Biomaterials 1992, 13, 67-97. (2) Guilak, F.; Kraus, V. B.; Setton, L. A. In Textbook of Sports Medicine; Garrett, W. E., Speer, K., Eds.; Williams and Wilkins: Baltimore, MD, 2000; pp 53-73. (3) Suh, H.; Lee, J. E. Yonsei Med. J. 2002, 43, 193-202. (4) Jay, G. D. Connect. Tissue Res. 1992, 28, 71-88. (5) Schmidt, T. A.; Gastelum, N. S.; Nguyen, Q. T.; Schumacher, B. L.; Sah, R. L. Arthritis Rheum. 2007, 56, 882-891.
(6) Englert, C.; McGowan, K. B.; Klein, T. J.; Giurea, A.; Schumacher, B. L.; Sah, R. L. Arthritis Rheum. 2005, 52, 1091-1099. (7) Klein, J. Proc. Inst. Mech. Eng., Part J 2006, 220, 691-710. (8) Mow, V. C.; Ateshian, G. A. In Basic Orthopaedic Biomechanics, 2nd ed.; Mow, V. C., Hayes, W. C., Eds. 1997; pp 275-315. (9) Hills, B. A. Proc. Inst. Mech. Eng., Part H 2000, 214, 83-94. (10) MacConaill, M. A. J. Anat. 1932, 66, 210-227. (11) McCutchen, C. W. Wear 1962, 5, 1-17. (12) Raviv, U.; Giasson, S.; Kampf, N.; Gohy, J. F.; Jerome, R.; Klein, J. Nature 2003, 425, 163-165. (13) Walker, P. S.; Dowson, D.; Longfiel.Md; Wright, V. Ann. Rheum. Dis. 1968, 27, 512-520. (14) Bell, C. J.; Ingham, E.; Fisher, J. Proc. Inst. Mech. Eng., Part H 2006, 220, 23-31. (15) Hills, B. A.; Butler, B. D. Ann. Rheum. Dis. 1984, 43, 641-648. (16) Schwarz, I. M.; Hills, B. A. Br. J. Rheumatol. 1996, 35, 821-827. (17) Schwarz, I. M.; Hills, B. A. Br. J. Rheumatol. 1998, 37, 21-26. (18) Jay, G. D. Curr. Opin. Orthop. 2004, 15, 355-359. (19) Jay, G. D.; Britt, D. E.; Cha, C. J. J. Rheumatol. 2000, 27, 594-600. (20) Jay, G. D.; Cha, C. J. J. Rheumatol. 1999, 26, 2454-2457.
10.1021/la702366t CCC: $40.75 © 2008 American Chemical Society Published on Web 01/09/2008
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Figure 1. Schematic representation of the 1404AA lubricin structure. N-terminal somatomedin B (SMB)-like domains and a C-terminal hemopexin (PEX)-like domain are separated by a large, central mucinlike domain.
the physicochemical nature of the cartilage surface and provide a chondroprotective function, likely by keeping the rubbing surfaces spaced apart. The glycoprotein, lubricin, and superficial zone protein (SZP) are thought to be the main components in synovial fluid and on the cartilage surface that provide this lubricating ability.4,18-27 Lubricin, a mucinous glycoprotein (Mw ≈ 240 kDa) first described by Swann,25-27 is a product of the gene proteoglycan 4 (PRG4), which is highly expressed by synoviocytes.19 Lubricin is an alternately spliced 1404 amino acid protein that is subject to extensive O-linked glycosylation (50% w/w) with NeuAc(R2,3)-Galβ(1,3)-GalNAc in its mucinlike central domain.21,28 A schematic representation of lubricin is shown in Figure 1. The abundance of negatively charged and highly hydrated sugars in this domain likely contributes to lubricin’s boundary lubrication properties by providing strong repulsion through steric and hydration forces.7,21,29 Lubricin in its native state can form intra- and intermolecular disulfide bonds because of the availability of cysteines in the C- and N-termini domains.30 This suggests that, like mucins,31 lubricin can associate by the free ends and form extended supramolecular structures. Hyaluronic acid (HA), also called hyaluronan, is a long, linear, unbranched high-molecular-weight (500-3800 kDa)32 anionic disaccharide consisting of alternating units of glucuronic acid β(1-3) and N-acetylglucosamine β(1-4).33 HA is a major constituent of synovial fluid with a concentration that ranges from 1 to 4 mg/mL in healthy individuals and decreases after joint injury and in arthritic disease to ∼0.1 to 1.3 mg/mL.5,34 HA has been recognized as one of the key molecules that impart normal synovial fluid with its remarkable rheological properties.7,35 In addition to its putative role in hydrodynamic lubrication, (21) Jay, G. D.; Harris, D. A.; Cha, C. J. Glycoconjugate J. 2001, 18, 807815. (22) Jay, G. D.; Hong, B. S. Connect. Tissue Res. 1992, 28, 89-98. (23) Jay, G. D.; Lane, B. P.; Sokoloff, L. Connect. Tissue Res. 1992, 28, 245-255. (24) Jay, G. D.; Tantravahi, U.; Britt, D. E.; Barrach, H. J.; Cha, C. J. J. Orthop. Res. 2001, 19, 677-687. (25) Swann, D. A.; Radin, E. L. J. Biol. Chem. 1972, 247, 8069-8073. (26) Swann, D. A.; Silver, F. H.; Slayter, H. S.; Stafford, W.; Shore, E. Biochem. J. 1985, 225, 195-201. (27) Swann, D. A.; Slayter, H. S.; Silver, F. H. J. Biol. Chem. 1981, 256, 5921-5925. (28) Elsaid, K. A.; Jay, G. D.; Warman, M. L.; Rhee, D. K.; Chichester, C. O. Arthritis Rheum. 2005, 52, 1746-1755. (29) Schaefer, D. B.; Wendt, D.; Moretti, M.; Jakob, M.; Jay, G. D.; Heberer, M.; Martin, I. Biorheology 2004, 41, 503-508. (30) Jones, A. R. C.; Gleghorn, J. P.; Hughes, C. E.; Fitz, L. J.; Zollner, R.; Wainwright, S. D.; Caterson, B.; Morris, E. A.; Bonassar, L. J.; Flannery, C. R. J. Orthop. Res.2007, 25, 283-292. (31) Bansil, R.; Stanley, E.; Lamont, J. T. Annu. ReV. Physiol. 1995, 57 635657. (32) Forsey, R. W.; Fisher, J.; Thompson, J.; Stone, M. H.; Bell, C.; Ingham, E. Biomaterials 2006, 27, 4581-4590. (33) Benz, M.; Chen, N.; Israelachvili, J. J. Biomed. Mater. Res. 2004, 71A, 6-15. (34) Balazs, E. A. In Disorders of the Knee; Helfet, A. J., Ed.; Lippincott Co.: Philadelphia, PA, 1974; pp 63-75. (35) Tadmor, R.; Chen, N. H.; Israelachvili, J. N. J. Biomed. Mater. Res. 2002, 61, 514-523.
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HA has been shown to decrease pain for arthritic patients upon injection in their joints, likely by acting as an anti-inflammatory agent.36 Although it is clear that such synovial fluid constituents contribute to boundary lubrication of apposing articular cartilage surfaces, the possibly synergistic contribution of these molecules and details of how they mediate force interactions between surfaces are still largely unknown. In this study, we examine how purified human lubricin, HA, and their mixture (at physiological concentration) interact with and mediate normal force interactions between chemically well-characterized model substrate surfaces. Our model substrates were functionalized with hydrophilic and hydrophobic self-assembled monolayers (SAMs) to mimic the chemistry of articular surfaces at their extremes. This approach was chosen to study the details of the conformational mechanics and adsorption behavior of lubricin and HA by avoiding the added complication of the chemical heterogeneity and compliance of cartilage surfaces. Here we present results from (1) normal force measurements conducted with colloidal probe microscopy between SAM model surfaces in the presence of human lubricin (25-400 µg/mL), HA (0.5-3.34 mg/mL) and in a mixture of (200 µg/mL lubricin and 3.34 mg/mL HA), and (2) adsorption measurements using surface plasmon resonance (SPR). Materials and Methods Lubricin Preparation. Lubricin was purified from human synovial fluid as described previously.21 A series of lubricin solutions (25-400 µg/mL) were prepared from stock solution by dilution with phosphate-buffered saline (PBS) solution (Gibco, 1× PBS, pH 7.4). The concentration range was chosen to bracket the physiological value of ∼200 µg/mL found in rabbit synovial fluid.28 Although this lubricin concentration is found for lubricin within synovial fluid, lubricin also accumulates on articular surfaces where a significantly higher working concentration may be present.37 Preparation of Colloidal Probes and Substrate Surfaces. Borosilicate glass microspheres (10 ( 1.0 µm diameter, series 9010, Duke Scientific Co., Palo Alto, CA) were glued with a two-part heat-hardening epoxy (Epo-Tek no. 377) or a one-part photocuring epoxy (Norland Optical Adhesive no. 81) onto commercially available AFM cantilevers (V-shaped Si3N4 cantilever with a typical spring constant of 0.58 N/m, 115 µm long and 600 nm thick, Veeco) using an XYZ micromanipulator (Signatone, S-926) under an optical microscope. The colloidal probe cantilevers were then coated with a 5 nm chromium adhesion layer followed by a 45 nm gold layer using e-beam evaporation under vacuum (CHA solution e-beam evaporator).38 Glass slides or cover slips were gold coated as described above. Before gold evaporation, the slides/cover slips were cleaned by immersion in piranha solution (1:3 v/v H2O2/H2SO4) at 80 °C for 10 min, followed by thorough rinsing with Milli-Q-grade water. Modified cantilever probes were imaged by scanning electron microscopy (SEM) to check for surface defects and contamination and to determine the probe diameter. Preparation of Self-Assembled Monolayers (SAMs). A selfassembled monolayer (SAM) of a hydroxyl- or methyl-terminated thiol was obtained by immersing a clean, gold-coated glass slide, glass cover slip, or colloidal microcantilever overnight in a 1 mM ethanolic solution of the thiol of interest. To prepare methyl- or hydroxyl-terminated surfaces, 1-octadecanethiol (98%) (Aldrich Chemicals Co. Milwaukee, WI) or 11-mercapto-1-undecanol (97%) (Aldrich Chemicals Co. Milwaukee, WI) was used, respectively. After incubation, the substrates and the cantilevers were washed (36) Goto, H.; Onodera, T.; Hirano, H.; Shimamura, T. Tohoku J. Exp. Med. 1999, 187, 1-13. (37) Schumacher, B. L.; Schmidt, T. A.; Voegtline, M. S.; Chen, A. C.; Sah, R. L. J. Orthop. Res. 2005, 23, 562-568. (38) Kaholek, M.; Lee, W. K.; Ahn, S. J.; Ma, H. W.; Caster, K. C.; LaMattina, B.; Zauscher, S. Chem. Mater. 2004, 16, 3688-3696.
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with copious amounts of ethanol, sonicated in ethanol for 1 min (with the exception of colloidal probes), and then rinsed again in ethanol to remove excess thiol. The substrates and microcantilevers were dried in a stream of dry nitrogen and stored until further use.38 Normal Force Measurements by Atomic Force Microscopy (AFM). A substrate modified with the SAM layer of interest was rinsed with Milli-Q-grade water and then dried in a stream of nitrogen prior to AFM measurements. Interaction forces were measured using an AFM (MultiMode with a low-noise AFM head, Nanoscope III controller, Veeco, Santa Barbara, CA). The cantilever normal spring constant, kn, was estimated from the power spectral density of the thermal noise fluctuations in air.39 The normal photodiode sensitivity was determined in PBS solution from the constant compliance regime on approach on a hard substrate. Force-distance measurements were obtained using a constant ramp size of 1000 nm and a frequency of 1.16 Hz (2.3 µm/s, unless noted otherwise) at room temperature. The normal forces were normalized by the measured colloidal probe radius, R. Force measurements were performed in the presence of human purified lubricin over a wide range of solution concentrations (25, 50, 100, 200, 300, and 400 µg/mL, diluted in PBS), HA (1.5-1.8 MDa, sodium salt from Streptococcus Sp., Biochemica; 0.5, 1.0, 1.5, 2.0, 2.5, 3, and 3.34 mg/mL in PBS), or a mixture of them (200 µg/mL lubricin and 3.34 mg/mL HA). The lubricin and HA concentrations for the mixture were chosen to mimic physiologically relevant concentrations found in synovial fluid.28,40 Force measurements were performed in about 50 µL of the solution of interest after allowing 15 min of thermal equilibrium. A minimum of 10 consecutive force-distance curves was recorded at each position, and at least 5 positions were measured on each sample. Forcedistance curves were averaged from at least 50 individual measurements. To assess the effect of contact force, dwell time (contact time), and scan velocity on the approach and retraction force profiles, experiments were conducted with different force trigger thresholds, wait times before reversal, and approach/retraction velocities. Fitting Force-Distance Curves. A significant problem with AFM force measurements on polymeric layers is the determination of absolute zero (i.e., layer compressibility is not accounted for, leading to an offset error). To interpret the measured repulsive interactions better and to extract estimates for the offset distance, force-distance curves were fitted, when appropriate, with the Alexander-de Gennes (AdG) model. Although this model describes the interaction between two polymer brush surfaces,41 it has also been employed successfully to describe the interaction of physisorbed polymer layers42 and lubricin on surfaces.43 Using the Derjaguin approximation to account for the spherical AFM probe interacting with a flat substrate surface, the Alexanderde Gennes model takes the following form44
D ) d + 2h
F(D) 16πkBTL 2L ) 7 R D 35s3
5/4
[( )
+5
(2LD )
7/4
- 12
]
(1)
where D is the separation distance, L is the equilibrium thickness of one brush layer, s is the average distance between grafting sites, R is the radius of the colloidal probe, kB is Boltzmann’s constant, and T is the absolute temperature. To more accurately fit the AdG model to the measured force-distance curves, the separation distance, D, was adjusted by adding an offset distance, 2h, to the measured distance, d, where (39) Hutter, J. L.; Bechhoefer, J. ReV. Sci. Instrum. 1993, 64, 1868-1873. (40) Balazs, E. A. The Physical Properties of Synovial Fluid and the Special Role of Hyaluronic Acid. In Disorders of the Knee; Lippincott: Philadelphia, PA, 1974. (41) de Gennes, P. G. AdV. Colloid Interface Sci. 1987, 27, 189. (42) Leckband, D.; Israelachvili, J. Q. ReV. Biophys. 2001, 34, 105-267. (43) Zappone, B.; Ruths, M.; Greene, G. W.; Jay, G. D.; Israelachvili, J. N. Biophys. J. 2007, 92, 1693-1708. (44) Kamiyama, Y.; Israelachvili, J. Macromolecules 1992, 25, 5081-5088.
(2)
This approach provides an offset distance, h, that enables the plotting of AFM force curves more correctly because it provides an estimate for the layer thickness at the maximal applied force in the measurement. Force curves were fitted individually with the AdG model, and average values of the fit parameters (L, h, and s) were calculated. The pressure was estimated from45 P(D) )
1 δF kT 2L ) 2πR ∂D s3 D
[( ) - (2LD ) ] 9/4
3/4
(3)
See Supporting Information for further details. Adsorption Measurements by Surface Plasmon Resonance (SPR). The adsorption of molecules onto chemically functionalized gold surfaces was measured by surface plasmon resonance using an SPR Biacore 1000 instrument (Biacore AB, Uppsala, Sweden).46,47 SPR substrates were prepared by first modifying gold-coated glass cover slips with hydroxyl- or methyl-terminated SAMs (as described above) and then mounting the substrates into an empty Biacore cassette using double-sided adhesive tape (3M Inc., St. Paul, MN). The blank SPR substrates were first equilibrated with PBS (pH ∼7.4), injected at a flow rate of 5 µL/min at 25 °C. A volume of 15 µL of the solution of interest (lubricin at a concentration ranging from 25 to 400 µg/mL and HA ranging from 0.5 to 3.34 mg/mL) was injected sequentially with increasing solution concentration over the functionalized gold surface. Each lubricant injection step was followed by a PBS rinse to remove loosely adsorbed molecules. The change in the response units (RU) before and after each injection is proportional to the number of molecules adsorbed at the surface (1 RU equals approximately 1 pg of protein/mm2). An adsorption isotherm can then be established by plotting the adsorbed amount as a function of solution concentration. SPR measurements are described in further detail in Supporting Information.
Results Adsorption of Lubricin and HA on Self-Assembled Monolayers. Typical adsorption isotherms (Figure 2a) show that lubricin adsorbs onto -CH3-terminated (hydrophobic) and -OHterminated (hydrophilic) surfaces with monolayer coverage at solution concentrations of about 200-300 µg/mL. These concentrations are typical physiological concentrations found in synovial fluid.18 Lubricin’s adsorption behavior is in stark contrast to that of HA, which adsorbed minimally onto both -CH3 and -OH surfaces (Figure 2b) even at solution concentrations 10 times larger than those used for lubricin. The average adsorbed amount (calculated from SPR measurements) at a solution concentration of 200 µg/mL lubricin, 3.34 mg/mL HA, and a mixture of the two on hydroxyl- and methylterminated SAM surfaces is shown in Figure 3. To assess the statistical relevance of the difference in adsorption in various solutions, analysis of variance (ANOVA) was performed, followed by Fisher’s least-significant-difference test. The results show that lubricin and the mixture adsorbed significantly more than HA on both the hydroxyl and methyl surfaces (p < 0.001). The mixture adsorbed more on the -CH3-terminated surface than did either of the constituents (p < 0.001). More lubricin consistently adsorbed onto -CH3-terminated surfaces than on -OH-terminated surfaces. Overall, more lubricants adsorbed on the hydrophobic methyl surface than on the hydrophilic hydroxyl surface (p < 0.0001). To further investigate the possibility of interaction between lubricin and HA, we injected an HA solution (3.34 mg/mL) on (45) Butt, H. J.; Cappella, B.; Kappl, M. Surf. Sci. Rep. 2005, 59, 1-152. (46) Advincula, R.; Aust, E.; Meyer, W.; Knoll, W. Langmuir 1996, 12, 35363540. (47) Jordan, C. E.; Corn, R. M. Anal. Chem. 1997, 69, 1449-1456.
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Figure 2. Change in SPR response units (∆RU) with respect to PBS baseline, plotted as a function of biopolymer solution concentration. (a) Lubricin and (b) HA adsorption isotherms on hydroxyl (-OH)- and methyl (-CH3)-terminated SAM surfaces. One RU corresponds to about 1 pg/mm2 of adsorbed biopolymer.
Figure 3. Average adsorbed amount (calculated from SPR data) at a solution concentration of 200 µg/mL lubricin, 3.34 mg/mL HA, and a mixture of the two on hydroxyl- and methyl-terminated SAM surfaces. The error bars represent the standard error of the mean of at least three independent measurements.
a preadsorbed lubricin layer (200 µg/mL). The HA injection did not lead to an increase in the adsorbed amount beyond that of the lubricin already present on the surface (Figure S4, Supporting Information). Normal Force Interactions. Approach CurVes. Although our AFM measurements were quite repeatable, we note that the conformational state of the physisorbed biopolymer layer likely does not reflect a thermodynamic equilibrium state (i.e., the repeated compression of the adsorbed polymer layer leads to a
Chang et al.
nonequilibrium segment density profile48,49). The details of the interaction thus depend on the experimental conditions, such as loading rate, contact force, and time in (dwell time) and out of contact.42 Lubricin. Averaged AFM normal force curves, measured upon approach between a flat surface and a colloidal probe (both modified with either a methyl- or hydroxyl-terminated SAM) for a range of lubricin concentrations, are shown in Figures 4a and 5a. The interactions in the presence of lubricin are monotonically repulsive, and the extent of the repulsion increases with increasing lubricin concentration. The measured forcedistance behavior on approach, above a minimum adsorbed amount of about 2.8 mg/m2, was reasonably well described by AdG theory. The surface coverage was estimated from the adsorption isotherms (Figure 2) at a solution concentration of 50 µg/mL on -CH3-terminated surfaces and 100 µg/mL on -OHterminated surfaces. The insets in Figures 4a and 5a show typical AdG fits to the data. These fits yielded estimates of the brush length, L, the distance between grafting sites, s, and the offset distance, h, shown in Figures 4b and 5b, for -CH3- and -OHterminated surfaces, respectively. The offset distance was used to more properly present the force-distance data obtained from AFM measurements by offsetting the normal force curve by 2h. The AdG model fit yielded a robust estimate of the extent of the “brush-like” steric repulsive interactions and the offset distance. We were unable to find alternative combinations of L, h, and s other than those obtained from the least-squares fitting approach that would provide an equally good or better fit to the data. We note that instead of fitting the average force-distance curves (shown in Figures 4a and 5a) with the AdG model we fitted each individual curve in a set and calculated the average values of the fit parameters. Figures 4b and 5b show that the average brush length and offset distance increase with increasing lubricin concentration while the apparent spacing between grafting sites remains essentially constant. In PBS and in the absence of lubricin, the interactions upon approach between each pair of SAM surfaces are quite different. On methyl-terminated SAM surfaces, the interaction is attractive (dashed line in Figure 4a) because of attractive hydrophobic interactions between the approaching surfaces. On hydroxylterminated SAM surfaces, the interaction is repulsive with a decay length of about 0.9 nm, in good agreement with the expected Debye length of about 0.8 nm for PBS (0.14 M NaCl + 2.7 mM KCl). Hyaluronic Acid (HA). Normal force interactions upon approach between methyl-terminated surfaces in the presence of HA can be grouped qualitatively into four types based on characteristic features of the approach curves (Figure 6): (A) a small steric repulsion, (B) a small steric repulsion at ∼15 nm distance followed by immediate jump-to-contact, (C) small jumpto contact, and (D) large jump-to-contact. The frequency with which each type was observed is listed for each HA concentration in Table 1. The greatest probability of a type A force curve occurs at the highest HA concentration (3.34 mg/mL), and the greatest probability of a type D force curve occurs at the lowest HA concentration (0.5 mg/mL). Averaged AFM normal force curves, measured upon approach between hydroxyl-terminated SAM surfaces, for a range of HA concentrations are shown in Figure 7a, and typical approachretraction curves for select HA concentrations are shown in Figure 7b. The data show that the interactions upon approach are (48) Klein, J.; Luckham, P. F. Macromolecules 1984, 17, 1041-1048. (49) Luckham, P. F.; Klein, J. Macromolecules 1985, 18, 721-728.
Lubricin and Hyaluronic Acid on Model Surfaces
Figure 4. (a) Force, normalized by probe radius, plotted as a function of separation distance measured upon approach between two methyl (-CH3)-terminated SAM surfaces for a range of lubricin concentrations. Each approach curve represents an average of at least 50 individual curves. A typical force curve in PBS is shown for comparison. The approach curves at a solution concentration of g50 µg/mL are offset by the offset distance, 2h, calculated from fits with the AdG model. (Inset) Typical AdG fit. (b) Brush length, L, offset distance, h, and spacing, s, obtained from fits with AdG plotted as a function of lubricin concentration. The error bar represents the standard deviation of the mean obtained from individual curve fits.
monotonically repulsive; however, compared with lubricin, the interaction distances are small. Lubricant Mixture (200 µg/mL Lubricin with 3.34 mg/mL HA). AFM normal force curves, measured upon approach between a methyl-terminated SAM surface and a methyl-terminated colloidal probe, are shown for three different lubricant solution concentrations and, for comparison, in PBS solution in Figure 8a. As before, the approach force curves showed a large jumpto-contact in PBS that is likely due to the hydrophobic attraction between the probe and the substrate surface. The force-distance curves measured in the lubricin/HA mixture showed, when compressed, the largest repulsion compared to those for HA or lubricin alone. Similar behavior was observed for interactions between hydroxyl surfaces (Figure 8b); however, the extent of the repulsive force for lubricin and the mixture was larger than on the -CH3-terminated surfaces (compare parts a and b of Figure 8), and the force between bare -OH-terminated surfaces in PBS was repulsive. Retraction CurVes. Lubricin. Although the interactions upon approach were, with the exception of the bare -CH3 surfaces, always repulsive, an adhesive interaction arose upon surface separation (i.e., the repulsion was not reversible). This hysteresis occurred on both -OH- and -CH3-terminated surfaces. A set of typical approach and retraction curves between -OHand -CH3-terminated surfaces is plotted in Figure 9 for a range of lubricin concentrations. At low lubricin concentrations, the
Langmuir, Vol. 24, No. 4, 2008 1187
Figure 5. (a) Force, normalized by probe radius, plotted as a function of separation distance measured upon approach between two hydroxyl (-OH)-terminated SAM surfaces for a range of lubricin concentrations. Each approach curve represents an average of at least 50 individual curves. A typical force curve in PBS is shown for comparison. The approach curves at a solution concentration of g100 µg/mL are offset by the offset distance, 2h, calculated from fits with AdG theory. (Inset) Typical AdG fit. (b) Brush length, L, offset distance, h, and spacing, s, obtained from fits with AdG theory plotted as a function of lubricin concentration. The error bar represents the standard deviation of the mean obtained from individual curve fits.
retraction curves showed features that are characteristic of repeated molecular stretching events. The magnitude and the extent of these interactions increase with increasing surface coverage to give way to large protracted adhesion forces. Histograms of the adhesion energy between hydroxyl- or methyl-terminated SAM surfaces, calculated from the area under the retraction curves for a range of lubricin concentrations, are shown in Figure 10a,b, respectively, which shows that the adhesion energy distributions broaden with increasing solution concentration. Figure 10c shows that the average adhesion energy increases monotonically and plateaus at solution concentrations between 200 and 300 µg/mL. The average adhesion energy measured between hydroxyl surfaces in the presence of high lubricin concentrations (>200 µg/mL) is about 2 times larger than that measured between methyl surfaces. Hyaluronic Acid (HA). The adhesive behavior between methylterminated surfaces in the presence of HA solutions is shown in Figure 6 (retract curves). Only in the case where the approach curves were monotonically repulsive (type A) was adhesion absent. For the three other types (B-D) showing attractive forces on approach, there were also large adhesive forces (pull-off forces) upon retraction (∼80 nN). These adhesive forces were larger than those between the hydrophobic surfaces in PBS. The adhesive interactions between hydroxyl-terminated surfaces in the presence of HA solutions were small (∼0.9 nN) when compared to those observed on the methyl surfaces (Figure S8, Supporting Information).
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Figure 6. Types of normal force curves (approach and retraction) obtained from methyl-terminated SAM surfaces in the presence of HA solution. (Inset) Force curve in pure PBS solution for comparison. Table 1. Percentages of Different Curve Types Obtained at Different HA Concentrationsa curve types
HA concentration (mg/mL)
A (%)
B (%)
C (%)
D (%)
0.5 1.0 1.5 2.0 2.5 3.0 3.34
30 35 42 68 61 48 70
8 2 10 32 33 26 24
8 9 33 0 6 9 6
55 53 15 0 0 17 0
a A, small steric repulsion; B, small steric repulsion at 15 nm distance followed by immediate jump-to-contact; C, small jump-to-contact; D, Large jump-to-contact.
Lubricant Mixture (200 µg/mL Lubricin with 3.34 mg/mL HA). The adhesive interactions upon retraction between methyland hydroxyl-terminated surfaces measured in the presence of the lubricin/HA mixture were similar to those observed between these surfaces in the lubricin solution (data not shown).
Discussion Adsorption of Lubricants. Our adsorption measurements show that lubricin is highly surface-active, adsorbing readily onto hydrophobic and hydrophilic surfaces. Additional experiments revealed that lubricin adsorbs even on surfaces functionalized with triethylene glycol-terminated SAMs (results not shown), which are typically resistant to protein adsorption.50 This amphipathic character allows lubricin to adsorb and to mediate surface interactions apparently independently of surface polarity. The estimated amounts of lubricin adsorbed on our model surfaces at solution concentrations of 200 µg/mL are 2.3 and 3.3 mg/m2 on the -OH- and -CH3-terminated surfaces, (50) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1167.
Figure 7. (a) Force, normalized by probe radius, plotted as a function of separation distance measured upon approach between two hydroxyl (-OH)-terminated SAM surfaces for a range of HA concentrations. Each approach curve represents an average of at least 50 individual curves. (b) Typical force-separation curves measured between hydroxyl-terminated SAM surfaces in the presence of 0.5, 2.5, and 3.4 mg/mL HA solutions.
respectively (i.e., more lubricin adsorbs onto -CH3- than onto -OH-terminated surfaces at the same solution concentration). These amounts are in surprisingly good agreement with the surface coverage of 2 mg/m2 found for lubricin/SZP on bovine menisci.37 The estimated surface mass densities at a solution concentration of 200 µg/mL yield an average spacing between adsorbed molecules of about 13 and 11 nm on the -OH- and -CH3terminated surfaces, respectively. These values are significantly smaller than the rough estimate of the radius of gyration of a 240 kDa glycoprotein with about 200 nm contour length and suggest that lubricin adopts an extended surface conformation.41 Moreover, these values compare favorably to the estimated spacing between grafting points, s, obtained from AdG model fits of about 16 and 20 nm on the -OH- and -CH3-terminated surfaces, respectively (Figures 4b and 5b).
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Figure 8. Force, normalized by probe radius, plotted as a function of separation distance measured upon approach between (a) methylterminated and (b) hydroxyl-terminated SAM surfaces in the presence of 200 µg/mL lubricin, 3.34 mg/mL HA, and a mixture of 200 µg/mL lubricin and 3.34 µg/mL HA. Each approach curve represents an average of over 50 individual curves. Typical force curves in PBS are shown for comparison. (Inset) AdG fit values for L, h, and s.
Figure 9. Typical force-separation curves measured between (a) methyl- and (b) hydroxyl-terminated SAM surfaces for a range of lubricin concentrations.
The facility with which lubricin adsorbs to hydrophilic and hydrophobic surfaces is not surprising given its molecular structure of largely hydrophobic globular domains in the N and C termini, separated by a heavily glycosylated linear region (mucin domain) that is negatively charged and hydrophilic (Figure 1).30 On hydrophobic surfaces, lubricin likely adopts a compact looplike conformation in which the hydrophobic ends serve as surface anchors; this allows the negatively charged and hydrated central mucin domain to extend away from the surface and interact more freely with water (Figure 10a). This surface conformation has been suggested for lubricin43 as well as for mucins on hydrophobic surfaces.51 On hydrophilic -OH-terminated surfaces, lubricin more likely adsorbs anywhere along its hydrophilic central domain, possibly through hydrogen bonds, and adopts, with increasing solution concentration, an extended tail-like conformation (Figure 11b). These adsorption behaviors are consistent with our normal force measurements (see the discussion below). Lubricin’s N- and C-terminal domains contain cysteines that form inter- and intramolecular disulfide bonds. This is a general molecular architecture that lubricin shares with that of mucins, a family of secreted glycoproteins that form protective and lubricous coatings on surfaces of various internal organs.31,51 We further note that the lubricin used in our study was not reduced
and alkylated and thus dimers, and possibly oligomers, were likely present in solution and on the surfaces.30 In contrast, only small amounts of HA adsorbed onto the hydrophobic and hydrophilic model surfaces even though the HA solutions were 10 times more concentrated than those of lubricin. This poor adsorption behavior of HA is consistent with results from previous studies using a surface force apparatus35,52 and has been suggested to be responsible for the lack of boundary lubrication properties.52 Whether HA alone is a good boundary lubricant is still unclear.5,14,32,35 However, several studies have suggested that HA interacts synergistically with lubricin to promote chondroprotection and adsorption.4,5,23 Our experiments showed an increase in the adsorbed amount on hydrophilic and hydrophobic surfaces when a mixture of lubricin and HA was presented to the substrate surface (Figure 3). The interaction between lubricin and HA in a mixture is weak and likely due to physical entanglement rather than a chemical interaction because the addition of HA to preadsorbed lubricin did not cause measurable HA adsorption (Figure S2, Supporting Information). Repulsion between Model Surfaces in the Presence of Lubricin and HA. Our normal force measurements show long-
(51) Lee, S.; Muller, M.; Rezwan, K.; Spencer, N. D. Langmuir 2005, 21, 8344-8353.
(52) Tadmor, R.; Chen, N.; Israelachvili, J. Macromolecules 2003, 36, 95199526.
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Figure 10. Histograms of adhesion energies (obtained from at least 100 force curves at each concentration) between (a) methyl- and (b) hydroxyl-terminated surfaces plotted for a range of lubricin concentrations. (c) Average adhesion energy measured upon retraction plotted as a function of lubricin concentration. The error bar represents the standard error of the mean.
range steric-entropic repulsion forces between physisorbed lubricin layers. These repulsion forces arise from the osmotic pressure that develops when the thermally mobile, physisorbed polymer chains on both surfaces are brought into increasingly stronger confinement. Predictably, the magnitude of the repulsion force increases with increasing lubricin concentration (i.e., surface coverage). Electrostatic interactions play only a secondary role because of strong charge screening. (The Debye length in PBS is on the order of 0.8 nm.) The measured force-distance behavior on approach was captured reasonably well by the AdG theory above a minimum adsorbed amount of about 2.8 mg/m2. The good agreement between the values for s, shown in Figures 4b and 5b, and the estimates obtained from the adsorption data (see above) lends further credibility to the use of AdG theory to describe the data. Although AdG theory was developed to describe the force interactions between high-molecular-weight, end-attached polymer coils at high grafting densities (brush behavior), it has also been used to describe the repulsion forces between surfaces exposing short-chained, flexible segments42 as well as the compressibility of physisorbed lubricin.43 This suggests that the AdG theory is able to capture the characteristics of a brush-like layer that arises from the adsorption of polymeric molecules in various conformations (tails, trains, and loops).
AFM force measurements are sensitive to the interactions between the distal ends of polymers emanating from the interacting surfaces. When comparing the overall shape of the force-separation behavior, particularly at large lubricin concentrations (compare Figures 4a and 5a), it becomes clear that lubricin is more compressible on -CH3-terminated surfaces than on -OHterminated surfaces (i.e., for a given force, the layer can be compressed more). It was found that for polydisperse polymer layers the force upon compression rises more gradually than in monodisperse layers and that effects of nonuniform chain length are most prominent at the periphery of a polymer brush layer and thus appear in the weak compression regime of the F/R(D) data.53 This could explain the difference in the compression behavior as seen in the F/R(D) data when comparing Figures 4a and 5a and would be consistent with the proposed adsorption behavior of lubricin. We suppose that lubricin adopts a packed, looplike structure on -CH3-terminated surfaces; however, there are likely still some chain ends and loops emanating far from the surface that can be compressed relatively easily. On -OH-terminated surfaces, however, lubricin adopts a more uniformly extended, homogeneous tail-like conformation, causing the force to rise more steeply upon compression. We note that the apparent extent of the lubricin tails and loops likely reflects a nonequilibrium (53) Patel, S. S.; Tirrell, M. Annu. ReV. Phys. Chem. 1989, 40, 597-635.
Lubricin and Hyaluronic Acid on Model Surfaces
Figure 11. Schematic representation of the proposed adsorption behavior of lubricin on (a) hydrophobic, methyl-terminated and (b) hydrophilic, hydroxyl-terminated SAM surfaces.
state because of the number and frequency of the repeated compression cycles in the AFM colloidal probe experiment. The overall lengths of the lubricin brush on -OH- and -CH3terminated surfaces, predicted from AdG theory, do not differ considerably, although the lengths are overall somewhat smaller on -CH3-terminated surfaces. The predicted lengths are consistent with scanning electron microscope observations by Swann et al. that suggest that single molecules of lubricin are about 200 nm long.27 Direct pressure measurements at the surface of the femoral head during free speed gait have shown pressures reaching around 500 kPa over most of the joint with a peak pressure of 5 MPa in some areas.54 The pressures that we achieved in our experiments were significantly smaller and ranged between 10 and 85 kPa (cf. eq 3 at the highest applied force). Our results, however, still provide useful information because typically more than 90% of the joint loading forces are supported by the liquid phase in the cartilage through interstitial fluid pressurization (IFP).8,55 Therefore, the pressures applied here approach those expected to occur between directly contacting cartilage surfaces (solid phase) under normal joint-loading conditions.8 The normal force interactions observed for HA on hydrophobic surfaces are consistent with the low HA adsorption measured by SPR. For example, the different types of normal force interactions upon approach, as depicted in Figure 6 (see also Table 1), may be explained by the difference in surface coverage with HA molecules. At high solution concentrations, there is some small and likely flat adsorption of HA, and the adsorbed molecules can thus maintain the surfaces separated by imparting repulsion forces, overcoming the strong hydrophobic attractive forces of the bare surfaces. It is not entirely clear why for hydrophobic surfaces at low HA solution concentrations the attractive interactions are (54) Morrell, K. C.; Hodge, W. A.; Krebs, D. E.; Mann, R. W. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 14819-14824. (55) Park, S. H.; Krishnan, R.; Nicoll, S. B.; Ateshian, G. A. J. Biomech. 2003, 36, 1785-1796.
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significantly magnified over the hydrophobic interactions of the bare surfaces. The strong jump-to-contact observed, for example, in type D force curves may be explained by the hydrophobic interaction between bare substrate surfaces, possibly aided by polymer bridging, because even at low HA solution concentrations there is some minimal HA adsorption. On hydroxyl surfaces, the HA provided weak steric repulsive forces at all solution concentrations, and adhesive interactions were often absent or small. Particularly at larger compression, the overall extent of the steric interactions in mixtures of lubricin and HA are slightly larger than those for lubricin alone (Figure 8). A mechanism that could explain this observation is that in the physical mixtures lubricin acts as the surface anchor for HA, leading to a larger mass density on the surface. This is consistent with the greater adsorbed mass observed for the HA/lubricin mixture compared with that for lubricin alone. The observed interaction between lubricin and HA here is likely due to a physical entanglement rather than chemical affinity because the presentation of HA to a previously adsorbed layer of lubricin did not lead to a measurable mass increase (Figure S2, Supporting Information). The ability of lubricin to adhere to a variety of surfaces and to form loops and tail-like layers that upon overlap can develop strong steric repulsion forces may be critical for lubricin to act as an effective antiadhesive and chondroprotectant on cartilage surfaces. Adhesion Forces between Model Surfaces in the Presence of Lubricin and HA. Although the potential role of lubricin in protecting articular surfaces from wear by causing strong repulsion forces is consistent with the present data, the interpretation of the effect of lubricin on adhesion between surfaces is more complicated. Adhesion was observed between lubricin layers adsorbed on both hydroxyl- and methyl-terminated surfaces. The characteristic shape of the retraction force curves (Figure 9) suggests that these adhesive interactions persist over long retraction distances, particularly at high lubricin concentrations. These adhesion forces likely arise from chain disentanglement and from stretching and breaking molecular bridges that form between molecules (through nonspecific interactions between chain segments) and between the interacting surfaces (through adsorbed molecules tethering the surfaces). This is most clear at low solution concentrations (i.e., below monolayer coverage), where apparently tethered molecules, likely bridging the two surfaces, were stretched. These bridges can form when at low surface coverage the adsorbed layers are incomplete and possibly deeply interpenetrated under confinement, causing the molecules to bind to both substrates at the same time. The interaction distance upon separation at high lubricin solution concentrations is often larger than twice the contour length of monomeric lubricin, suggesting the presence of dimers and possibly oligomers linked by disulfide bridges. Furthermore, our experiments were all carried out in lubricin solutions, and thus, particularly at high solution concentrations, the adhesive interactions upon separating the lubricin-covered surfaces may involve contributions from the untangling of lubricin molecules. As shown in Figure 10c, the average adhesion energy, obtained by averaging the areas under the retract portion of a set of force curves, increases monotonically and plateaus for a lubricin solution concentration between 200 and 300 µg/mL. This functional behavior is similar to that observed for the adsorption and suggests that adhesive interactions reach a plateau at about monolayer surface coverage. This lends further support to an adhesion mechanism involving bridging interactions between the separating surfaces. Furthermore, the average adhesion energy
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Figure 12. Schematic representation of the steric and bridging interaction behaviors of lubricin on hydrophobic, methyl-terminated (a, c) and hydrophilic, hydroxyl-terminated (b, d) SAM surfaces at low (a, b) and high surface coverage (c, d).
was higher on hydroxyl surfaces than on methyl surfaces. This behavior is consistent with the proposed adsorption and conformation behavior of lubricin. Bridging may occur more easily on hydroxyl-terminated surfaces for several reasons. Lubricin adsorption likely occurs through interactions with segments in the large central mucin domain, and under confinement, segments are likely to adsorb on both surfaces. Furthermore, we suppose that lubricin adopts a conformational state in which nonpolar chain ends emanating from one surface can interact hydrophobically with those emanating from the opposing surface. Finally, it is likely that fewer loops are formed on hydroxyl surfaces than on methyl-terminated surfaces, possibly allowing for easier chain entanglement (Figure 12). We note that the observed adhesion interactions reflect a nonequilibrium state and depend on the loading and unloading rates, the time in contact, and the applied pressure.42 To illustrate the effect of contact force, scan velocity, and dwell time (contact time) on the adhesion energy, we have conducted a set of exploratory experiments on -CH3-terminated surfaces at a constant lubricin solution concentration of 200 µg/mL. The effect of dwell time (i.e., time delay while in contact at the maximal force) is dramatic. Figure 13a,b shows that the average adhesion energy increases substantially with increasing time in contact. Figure 13c,d shows the effect of contact force and scan velocity on adhesion energy. Adhesion increases slightly with increasing contact force and decreases with decreasing scan velocity. The latter observation most likely reflects the overall shorter contact time that occurs at higher rates. Taken together, these observations are consistent with the notion that while in contact adsorbed
Figure 13. (a) Representative force curves measured between methyl-terminated surfaces as a function of dwell time. Adhesion energy measured upon retraction as a function of (b) dwell time, (c) contact force, and (d) scan velocity on methyl-terminated SAM surfaces. (Adhesion measurements discussed in the article were obtained with zero dwell time, 2.33 µm/s scan velocity, and 13-20 nN contact force, indicated by the arrows and bracket.)
lubricin layers appear to develop significant molecular interdigitations that effectively increase the molecular contact area and may also cause increased bridging interactions.42
Lubricin and Hyaluronic Acid on Model Surfaces
Conclusions We have shown that lubricin, even at relatively low surface concentrations, mediates normal force interactions between chemically well-defined model surfaces. Lubricin is amphiphilic and adsorbs onto both methyl- and hydroxyl-terminated surfaces, where it adopts quite different conformations. On hydrophobic surfaces, lubricin is in a compact, looplike conformation in which its hydrophobic domains at the N and C termini possibly serve as surface anchors. On hydrophilic surfaces, lubricin likely adsorbs along its hydrophilic, mucin-like central domain and adopts, with increasing solution concentration, an extended, tail-like conformation. On both surfaces, lubricin develops strong repulsive interactions. Furthermore, on surface separation, adhesion occurs between the surfaces, likely as as result of chain disentanglement and molecular bridging. This behavior is in contrast to that of HA, which does not adsorb appreciably on the model surfaces or develop significant steric repulsive interactions. Adhesive forces, particularly between the hydrophobic surfaces, are large and not appreciably mediated by HA. The extent of repulsive interactions on approach in physical mixtures of lubricin and HA is slightly larger than that for lubricin alone, consistent with
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the greater adsorbed mass observed for the HA/lubricin mixture compared with that for lubricin alone. Our results do not, however, suggest significant synergistic contributions to the normal force interactions. On the basis of these observations, we speculate that the ability of lubricin to adhere to a variety of surfaces and to form loops and tail-like layers that upon overlap can develop strong repulsive interactions may be critical for lubricin to act as an effective antiadhesive and chondroprotectant on cartilage surfaces, thereby preventing or reducing wear. Acknowledgment. This research was supported by an NSF Early Career Award (S.Z.), NIH grants AR50245 and AG15768 (F.G.), and AR050180 from NIAMS (G.D.J.). We thank Jeff Coles for many useful discussions and anonymous reviewers for informative discussions and perspectives. Supporting Information Available: Adsorption measurements using surface plasmon resonance, steric forces, and additional results. This material is available free of charge via the Internet at http://pubs.acs.org. LA702366T
